11, 15-dioxygenated progesterones



United States Patent O 11,15-DIOXYGENATED PROGESTERONES Milton N. Doniu,Metuchen, Josef Fried,.New Brunswick,

and Richard W. Thoma, Some Olin Mathieson Chemical Corporation, NewYork, N.Y., a corporation of Virginia No Drawing. Application December5, 1956 Serial No. 626,318

4 Claims. (Cl. 260-397.3)

This invention relates to, and has for its object the provision of, thenew steroids, 11,15B-dihydroxyproges terone (A pregnenel1a,15/3-dlOl-3,20-dl0116), the di-' esters thereof and11,15-diketoprogesterone (A -pregnene- 3,1 1,15,20 tetraone).droxyprogesterone is prepared by subjecting progesterone to the actionof enzymes of the microorganism Aspergillus giganteous under aerobicconditions. The hydroxylation can best be effected by either includingthe steroid in an aerobic culture of the microorganism, or by bringingtogether in an aqueous medium the steroid, air and enzymes ofnon-proliferating cells of the microorganism.

In general, the conditions of culturing Aspergillus giganteus for thepurposes of this invention are (except for the inclusion of the steroidto be converted) the same as those of culturing various other aerobicfungi for the production of antibiotics, organic acids or vita mins,i.e., the microorganism is aerobically grown in contact with (in or on)a suitable fermentation medium. A suitable medium essentially comprisesa source of nitrogenous factors and a source of carbon and energy. Thelatter may be a carbohydrate (such as sucrose, molasses, glucose,maltose, starch or dextrin), a higher fatty acid, a fat and/ or thesteroid itself. Preferably, however, the medium includes an assimilablesource of carbon and energy in addition to the steroid. The sources ofnitrogenous factors may be organic (e.g., soybean meal, corn steepliquor, meat extract and/or distillers solubles) or synthetic (e.g.,composed of simple, synthesizable organic and inorganic compounds, suchas ammonium salts, alkali nitrate, amino acids or urea).

An adequate sterile-air supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated cultures. Thesteroid may be added to the culture during the incubation period orincluded in the medium prior to sterilization or inoculation.

The process yields 11a,lSfl-dihydroxyprogesterone, which in turn caneither be esterified in the usual manner, as by treatment with an acylhalide or acid anhydride to yield the diester derivative, or oxidized asby treatment with a hexavalent chromic compound (e.g., chromic acid) toyield 11,15-diketoprogesterone. Although any acylating agent may be usedto form the diester, the preferred compounds are the acyl chlorides oracid anhydrides of hydrocarbon carboxylic acids having less than tencarbon atoms, as exemplified by the lower alkanoic acid anhydrides(e.g., acetic anhydride), monocyclic aryl carbonyl chlorides (e.g.,benzoyl chloride), monocyclic aralkanoic acid chlorides (e.g.,phenacetyl chloride), the lower alkenoic acid anhydrides and themonocycloalkanecarbonyl halides.

rville, N.J., assignors to The compound 11a,15;3 -'dihy 2,881,189Patented Apr. .7, 1959 ICC ' The steroids thus formed are of the generalformula wherein R is hydrogen, R is hydroxy' or acyloxy (particularlythe acyloxy radicalof a hydrocarbon carboxylic acid having less than tencarbon atoms) or together R and R is keto. v

11,15-diketoprogesteroneand the diesters'of :,155- dihydroxyprogesteroneare physiologically active steroids which possess progestationalactivity ,and, hence, can be used in' lieu of known'proge'stationalsteroids such as progesterone in the treatment of functional uterinebleeding, being formulated for such administration in the same type ofpreparations as progesterone, for example, with concentration and/ordosage based on the activtiy of the steroid.

The following examples illustrate the invention (all temperatures beingin centigrade):

EXAMPLE 1 11a,1Sfi-dihydroxyprogesterone (a) FERMENTATION The surfacegrowth from a two-week-old Sabourauddextrose agar slant of Aspergillusgiganteus ATCC 10059 (American Type Culture Collection, Washington, DC.)is transferred to 6 flasks, SOD-m1. Erlenmeyer type, each with 100 ml.of medium composed of 36 g. corn steep liquor, 10 g. brown sugar, 6 g.NaNO 1.5 g. KH PO 0.5 g. MgSO .7H O, 1 mg. ZnSO.,, 5 g. CaCO 2 g. lardoil and distilled water to 1 liter, sterilized by autoclaving at C. for40 min. The inoculated flasks are incubated at 25 C. and rotatedmechanically during incubation at 280 r.p.m. in a Z-inch radius. After48 hours, a 10% transfer is made to 30 similar flasks containing l00-ml.portions of medium composed of 6 g. corn steep liquor, 3 g. NH H PO,,2.5 g. CaCO 2.2 g. soybean oil,

0.25 g. progesterone and distilled water to 1 liter, sterilized byautoclaving at 120 C. for 30 min. Incubation of cultures with steroid isfor 68 hours. The contents of the flasks are pooled and filtered througha Seitz clarifying pad. Flasks and pad are washed with water. The volumeof filtrate and washings, representing 750 mg. of original steroid, is2750 ml.

(b) ISOLATION Ark, 242 my (e=l6,l00); A221? 3.03, 5.89, 6.06

3 4 Aruzlysis.Caled. fo r C H O (346.45% 2, 72.80; H, Analysis.---Calcd;,rbr;c; H ,'o;(342.42) C, 73;66; H, 8.73. =Foiind': C 72.88; H, 8.58.7.65. Found: C, 74.10; H, 7.82.

w h. r...'1he...invention--may-be otherwise variouslyembodied EXAMPLE 2within the scope of the appended claims. 11a,15,8-dihydroxyprogesteronedzacetate We claim: A solution of mg. of 11a,1S/S-dihydroxyprogesterone1 A steroid selected from the group consisting of in 1 ml. of anhydrouspyridine and 1 ml. of acetic an- 11u,15fi-dihydroxyprogesterone, 11,15-diketoprogesterhydride is allowed to remain at room temperature forone, and the 11 ,15;3-diester of 11a,15;3-dihydroxyprogeshours. Afterremoval of the reagents by evaporation terone with a hydrocarbonmonocarboxylic acid having ...invacuo,.there:remainsoa-residueiconsisting of '11a,15;8- 10 less than tencarbon atoms.

dihydroxyprogesterone diacetate. 2. 1101,15 fi-dihydroxyprogesterone.

EXAMPLE 3 3. 11a,1Sfl-dihydroxyprogesterone diacetate.

. 4. LI 1,15-dike1oprogesterone 11 15 'dlketopmgesterone 53 mg. of1'1u,1SB-dihydroiryprogesterone is dissolved 15 in 3 ml. offlglacialacetic acid and oxidized with a solu- References Cited m'thefilcio'f'tmspatcnt tion of mg'.--of-chromicsacid in 6.5 ml. of aceticacid. UNITED STATES, 'y After 30 minutes 0.5m. of methanol is added andthe mixture concentrated-to small volume. The mixture is 2,602,760"Murray 1952 taken up in chloroform and water and the chloroform 202,658,023 Shull Nov. 3, 1953 solution extracted with water, dilutebicarbonate and 2 703 05 Man 3 1955 ,agalmwlth W f f 95 2,753,290 FriedJuly 3, 1956 sodiumsulfate and-the solvent'removed m'vacuo. Theresidue-'(aboht"34.-3 mg:) is recrystallized from acetone- 2799'689 Fned1957 '1"- --"following"properties:-=M.P; about 1 915- ['a] 5 3+276 etherand affords pure 11,15-diketoprogesterone*'of the 25 OTHERJREFERENCES('c.,-"0.80 in-CHCI -Von"0.-Schindler: Helv. Chim. Actapvol. '39; N0. 2,79,53,238 Imp-(cameo);xgaifsfimaso,5298;605,6.16, Pages 375-394, 1956-

1. A STEROID SELECTED FROM THE GROUP CONSISTING OF11A,15B-DIHYDROXYPROGESTERONE, 11,15 -DIKETOPROGESTERONE, AND THE11A,15B-DIESTER OF 11A,15B-DIHYDROXYPROGESTERONE WITH A HYDROCARBONMONACARBOXYLIC ACID HAVING LESS THAN TEN CARBON ATOMS.